Shikimic acid is an optically active substance having three chiral carbons in its molecule, and is used as a raw material for the synthesis of a large number of medicines, agrichemicals, cosmetics, and the like. In particular, shikimic acid is known to be an important starting material for the chemical synthesis of Tamiflu (registered trademark), a medicine to treat influenza. Recently; the efficacy of Tamiflu for avian influenza, of which the pandemic is a great concern, has been demonstrated, and for this reason, there is a growing demand for shikimic acid as a raw material of Tamiflu. Also, shikimic acid can be chemically converted into useful chemicals, such as p-hydroxybenzoic acid and phenol, and is promising as a raw material for the synthesis thereof.
Shikimic acid has conventionally been obtained by extraction from the fruits of plants, such as Illicium anisatum and Illicium verum. However, the extraction and purification methods are complicated and of low yield, and in addition, since the raw materials are natural products, it is difficult to stably supply a large amount thereof.
Meanwhile, shikimic acid is an important intermediate in the aromatic compound biosynthetic pathway of bacteria, yeasts, plants, etc., and can be produced by fermentation using microorganisms having this pathway. Production of shikimic acid using Escherichia coli as a host has been reported so far (Patent Literature 1 to 7), but in the methods, quinic acid produced as a by-product together with shikimic acid is a factor hindering the purification of shikimic acid. Further, since shikimic acid is produced in association with aerobic growth in each method, glucose, which is a raw material of shikimic acid, is largely used for bacterial growth, resulting in low yield of shikimic acid as the objective substance. For example, the shikimic acid yield described in Patent Literature 4 and 6 is as low as 27%. The maximum yield of shikimic acid from glucose described in Patent Literature 1 is 43%, but here the possibilities of reproduction of phosphoenol pyruvate from pyruvic acid and glucose uptake by non-phosphotransferase system, etc. are not taken into consideration. Therefore, the actual maximum theoretical yield of shikimic acid from glucose is considered to be 86%. When this theoretical yield is used as a baseline, the above sugar-based yield of shikimic acid 27% is calculated to be 31% of the theoretical yield, which is also low.
Patent Literature 8 reports shikimic acid production using a mutant strain of Citrobacter freundii, the mutant strain having resistance to 4-hydroxy-3-methoxybenzoic acid as a 4-hydroxybenzoic acid analog. However, regarding the mutant strain, the mutation site is unknown, the concentration of shikimic acid produced is low, and the sugar-based yield is also unknown.
Patent Literature 9 and 10 report shikimic acid production using an aromatic amino acid auxotroph of Bacillus subtilis, but regarding the auxotroph, the mutation site is unknown, the concentration of shikimic acid produced is low, and the sugar-based yield is also unknown.